- Short genome report
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Complete genome sequence of the sand-sediment actinobacterium Nocardioides dokdonensis FR1436T
Standards in Genomic Sciences volume 12, Article number: 44 (2017)
Nocardioides dokdonensis, belonging to the class Actinobacteria, was first isolated from sand sediment of a beach in Dokdo, Korea, in 2005. In this study, we determined the genome sequence of FR1436, the type strain of N. dokdonensis, and analyzed its gene contents. The genome sequence is the second complete one in the genus Nocardioides after that of Nocardioides sp. JS614. It is composed of a 4,376,707-bp chromosome with a G + C content of 72.26%. From the genome sequence, 4,104 CDSs, three rRNA operons, 51 tRNAs, and one tmRNA were predicted, and 71.38% of the genes were assigned putative functions. Through the sequence analysis, dozens of genes involved in steroid metabolism, especially its degradation, were detected. Most of the identified genes were located in large gene clusters, which showed high similarities with the gene clusters in Pimelobacter simplex VKM Ac-2033D. Genomic features of N. dokdonensis associated with steroid catabolism indicate that it could be used for research and application of steroids in science and industry.
Bacteria in the genus Nocardioides were first isolated from soil in 1976  and currently more than 90 validly published Nocardioides species are available from diverse terrestrial and aquatic environments such as soil, wastewater, plant roots, groundwater, beach sand, and marine sediment [2,3,4,5,6,7,8,9,10]. Originally, the genus was classified as a member of the order Actinomycetales in the phylum Actinobacteria , but recently was reclassified to the order Propionibacteriales . Actinobacteria , also called Gram-positive high G + C bacteria, contain diverse bacterial groups that are capable of a variety of secondary metabolism including biosynthesis of antibiotics and degradation of harmful compounds [12, 13]. The genus Nocardioides is also known to utilize several kinds of non-degradable materials such as alkane compounds , atrazine , phenanthrene , trinitrophenol , and vinyl chloride . Despite almost 100 species with validly published names and their useful features associated with secondary metabolism, only draft genome sequences are publically available for the genus besides that of Nocardioides sp. JS614.
N. dokdonensis was isolated from beach sand in Dokdo, a volcanic island located in the East Sea of Korea, in 2005 . The East Sea is called a “mini-ocean” due to its oceanological properties  and is known to have a high microbial diversity . To reveal distinguishing genomic features of Nocardioides species, we determined and analyzed the genome sequence of N. dokdonensis FR1436T.
Classification and features
Nocardioides dokdonensis FR1436T, a Gram-positive, non-motile, and strictly aerobic bacterium, was isolated from sand sediment of the Dokdo island in Korea . The strain grows at the temperature range of 4 to 30 °C (optimum, 25 °C), pH range of 5.0 to 10.0 (optimum, 7.0), and NaCl concentration of 0 to 7% (w/v) (optimum, 0 to 3) . Its colony size is about 1.0–2.0 mm on TSA medium after incubation for 3 days at 25 °C. Cells are 1.2–1.8 μm long and 0.6–0.9 μm wide in size  (Fig. 1). FR1436 can utilize adonitol, glycerol, melezitose, melibiose, ribose, sodium acetate, sodium citrate, sodium propionate, and sodium pyruvate as a sole carbon source . Minimum information about the genome sequence (MIGS) for FR1436 is described in Table 1.
Phylogenetically, N. dokdonensis belongs to the family Nocardioidaceae of the order Propionibacteriales , and a phylogenetic tree based on the 16S rRNA genes of the type strains in the genus Nocardioides shows that N. dokdonensis FR1436 forms a sister clade with N. lianchengensis (Fig. 2), which was isolated from soil, and shares common ancestor with N. marinisabuli , N. basaltis , and N. salaries.
Genome sequencing information
Genome project history
As part of the project that investigates the genomic and metabolic features of bacterial isolates in and around Dokdo, the genome sequencing and analysis of N. dokdonensis FR1436 were performed at the Laboratory of Microbial Genomics and Systems/Synthetic Biology at Yonsei University. The complete genome sequence of N. dokdonensis FR1436T (= KCTC 19309 T = JCM 14815 T) has been deposited in GenBank under the accession number CP015079. The Bioproject accession number is PRJNA191956. A summary of the genome project is provided in Table 2.
Growth conditions and genomic DNA preparation
N. dokdonensis FR1436 was streaked on trypticase soy agar medium (Difco, 236,950) and incubated at 25 °C for 3 days. A single colony was inoculated in trypticase soy broth and incubated at 25 °C for 2 days. Cells in the exponential phase were harvested and genomic DNA was extracted using Wizard Genomic DNA Purification Kit (Promega, USA) according to the manufacturer’s protocol.
Genome sequencing and assembly
Genome sequencing of N. dokdonensis FR1436 was performed using the PacBio RS II System (Macrogen, Inc., Republic of Korea). A 20-kb library and C4-P6 chemistry were used for the genome sequencing. A total of 200,435 continuous long reads and 1,551,246,448 base pairs were generated after genome sequencing and quality trimming of the sequencing reads. De novo assembly was conducted with SMRTpipe HGAP and scaffolding and gap filling were performed with SMRTpipe AHA. Finally, consensus sequences were generated with SMRTpipe Quiver.
Structural gene prediction and functional annotation were conducted using the Prokka program . Additionally, we performed a functional assignment of the predicted protein-coding sequences using blastp against Pfam, Uniref90, KEGG, COG, and GenBank NR databases for more accurate annotation. tRNAscan-SE  and RNAmmer  were used for prediction of transfer RNAs and ribosomal RNAs, respectively. Assignment of the Clusters of Orthologous Groups was conducted with RPS-BLAST against COG database with an e-value cutoff of less than 1e-02. Clustered regularly interspaced short palindromic repeats were predicted with CRISPR Finder . Proteins containing signal peptide and transmembrane helices were predicted using SignalP  and TMHMM , respectively. Secondary metabolite biosynthetic genes were predicted using AntiSMASH program .
N. dokdonensis FR1436 has a single chromosome of 4,376,707 bp in length, and consists of 72.26% of G + C content (Fig. 3 and Table 3). The genome has 4165 genes that are comprised of 4104 CDSs, three rRNA operons, 51 tRNAs, and one tmRNA. Results from the analysis of KEGG pathways indicated that, in the genome of FR1436, all of the genes involved in glycolysis, gluconeogenesis, and citrate cycle are present and well conserved. Among the predicted genes, 71.38% of the genes were assigned putative functions and 2832 CDSs was functionally assigned to the COG categories (Table 4). Also in the genome, ten putative CRISPR repeats were predicted using the CRISPRFinder program, but there were no CRISPR-associated proteins next to the predicted repeat sequences. Two gene clusters, possibly associated with secondary metabolism, were predicted using the AntiSMASH program. One cluster (accession numbers ANH38050 to ANH38087) has genes associated with the phenylacetate catabolic pathway  and another cluster (accession numbers ANH40163 to ANH40204) has genes of type 3 polyketide synthases.
Insights from the genome sequence
In the genome of N. dokdonensis FR1436, dozens of steroid-degrading genes were detected (Additional file 1). Major functions of steroids, essential biomolecules in living organisms, include maintaining membrane fluidity as a component of the cell membrane and controlling cell metabolism as signaling molecules . Moreover, steroid medicines are used for treatment of a number of diseases from inflammation to cancer . The molecular backbone of steroids is composed of three cyclohexanes and one cyclopentane. To the backbone, diverse side chains are attached to endow them with diverse functions . Catabolic pathways of steroid degradation or modification have been analyzed in depth for some genera in the order Corynebacteriales [33,34,35]. In Nocardioidaceae , several large gene clusters, which have potential binding sites of the transcriptional regulator associated with steroid catabolism in their promoters, were predicted in the genome of Pimelobacter simplex VKM Ac-2033D . In the genome of FR1436, gene cluster A, which is known to be involved in degrading steroid rings A/B, and gene cluster B, which is involved in degrading side chains, were detected (Fig. 4). However, in FR1436, cluster A is separated into two large gene clusters and an additional mce gene cluster, which is involved in steroid uptake , was detected (Additional file 1). In VKM Ac-2033D, cluster A is located approximately 350-kb downstream of cluster B, whereas in FR1436, cluster A is located 6 kb downstream. Moreover, two kstR and 11 kstR2 genes, which encode the TetR family of transcriptional regulators and are reported to regulate cholesterol metabolism in mycobacteria , were detected (Additional file 1). Besides the genes in clusters A and B, genes encoding 3-beta-hydroxysteroid dehydrogenase (ANH36717 and ANH37882), 3-alpha-hydroxysteroid dehydrogenase (ANH37023 and ANH37488), and steroid delta-isomerase (ANH36955) were also detected in the genome of FR1436. Additionally, all genes involved in degradation of cholesterol to HIP-CoA were identified (Fig. 5). These results indicate that the genus Nocardioides can be useful for research and utilization of steroid metabolism.
Steroids are important biomolecules in living organisms and carry out diverse roles as components of the cell membrane to signaling molecules . Moreover, steroids are being used to treat various diseases from inflammation to cancer . These indicate that research on modification of steroid compounds has infinite possibilities to improve human health. To date, studies on bacterial steroid metabolism have been mainly focused on the order Corynebacteriales [33,34,35]. Recently, genome analysis of the genus Nocardioides in the order Propionibacteriales revealed several kinds of gene clusters associated with steroid degradation . In this study, we determined the complete genome sequence of N. dokdonensis FR1436 and analyzed the genome sequence to detect the presence of genes related to steroid metabolism. In the genome of FR1436, dozens of genes associated with steroid catabolism were detected in large gene clusters. These results demonstrate that bacteria in the genus Nocardioides can be used as promising candidates for steroid research and related fields of industry.
Basic Local Alignment Search Tool
Clusters of Orthologous Groups
Clustered regularly interspaced short palindromic repeat
Kyoto Encyclopedia of Genes and Genomes
Minimum information about a genome sequence
Reversed position specific-BLAST
UniProt reference clusters
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We recognize Ju Yeon Song for her involvement during the early stages of the project.
This work was financially supported by the National Research Foundation (NRF-2014M3C9A3068822 and NRF-2011-0017670) of the Ministry of Science, ICT and Future Planning, Republic of Korea.
The authors declare that they have no competing interests.
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Kwak, MJ., Kwon, SK. & Kim, J.F. Complete genome sequence of the sand-sediment actinobacterium Nocardioides dokdonensis FR1436T . Stand in Genomic Sci 12, 44 (2017). https://doi.org/10.1186/s40793-017-0257-z
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